SCF and the cullin-RING ubiquitin ligases (CRLs) comprise the largest family of ubiquitin ligases in humans. These ~250 enzymes have a major impact on human biology through their ability to specify the half-lives of key regulatory proteins that control processes from basic metabolism to circadian rhythms. In keeping with their broad impact on biology, CRLs also play a major role in human health and cancer chemotherapy. Substrate specificity of CRLs is determined by an interchangeable substrate receptor subunit (SRS). Several CRL SRSs, including Vhl and Fbxw7, are prominent tumor-suppressors, whereas others (Skp2) are proto- oncogenic and yet another, Crbn, is targeted by the drugs that are most commonly used to treat multiple myeloma. Moreover, Nedd8 conjugation, which regulates CRL assembly and activity, is the target of a cancer drug in clinical development. Given their extraordinary impact on human biology and health, CRLs have been studied intensively. However, there is a great deal that we still do not understand about these enzymes. For example, we do not fully understand how exchange of SRSs occurs and we do not know how the exchange process is regulated to ensure that cells possess an appropriate repertoire of CRL complexes. Even very basic questions remain unaddressed, such as: what is the fraction of SRSs that assemble to form CRLs? Do all SRSs assemble with equal efficiency?; do all cells express the same repertoire of CRL complexes?; and so forth. This application proposes development of new assays and methodologies that will enable us to address these fundamental questions. The first two Aims investigate the steady-state structure and dynamics of the cellular network of SCF complexes through development and application of a set of `Selected Reaction Monitoring' (SRM) mass spectrometry assays to identify and quantify all known SCF subunits and regulators. The third Aim focuses on development and application of new assays to monitor the dynamic association of the SRS exchange factor Cand1 with SCF. FRET and single molecule approaches will explore how Cand1 promotes SRS exchange, and how this process is regulated. Understanding the regulation of CRL assembly will reveal how the network of CRLs is controlled in normal and diseased cells.